Biomedical Engineering Reference
In-Depth Information
protein. This technique is called
south-western
screen-
ing, because it combines the principles of Southern
and western blots. It has been particularly successful
in the isolation of clones expressing cDNA sequences
corresponding to certain mammalian transcription
factors (Singh
et al
. 1988, Staudt
et al
. 1988, Vinson
et al
. 1988, Katagiri
et al
. 1989, Williams
et al
. 1991,
Xiao
et al
. 1991). A limitation of this technique is that,
since individual plaques contain only single cDNA
clones, transcription factors that function only in the
form of heterodimers or as part of a multimeric com-
plex do not recognize the DNA probe and the cor-
responding cDNAs cannot be isolated. Clearly the
procedure can also be successful only in cases where
the transcription factor remains functional when
expressed as a fusion polypeptide. It is also clear that
the affinity of the polypeptide for the specific DNA
sequence must be high, and this has led to the prefer-
ential isolation of certain types of transcription fac-
tor (reviewed by Singh 1993). More recently, a
similar technique has been used to isolate sequence-
specific RNA-binding proteins, in this case using a
single-stranded RNA probe. By analogy to the above,
this is termed
north-western screening
and has been
successful in a number of cases (e.g. see Qian &
Wilusz 1993; reviewed by Bagga & Wilusz 1999).
Both south-western and north-western screening are
most efficient when the oligonucleotide contains the
binding sequence in multimeric form. This may mean
that several fusion polypeptides on the filter bind to
each probe, hence greatly increasing the average
dissociation time. To minimize non-specific binding,
a large excess of unlabelled double-stranded DNA
(or single-stranded RNA) is mixed with the specific
probe. However, it is usually necessary to confirm
the specificity of binding in a second round of screen-
ing, using the specific oligonucleotide probe and
one or more alternative probes containing a similar
sequences that are not expected to be recognized.
on the preservation of the appropriate interacting
domain of the protein when exposed on the surface
of a nitrocellulose filter. Furthermore, as discussed in
Chapter 9, the yeast two-hybrid system and its
derivatives now provide versatile assay formats for
many specific types of protein-protein interaction,
with the advantage that such interactions are tested
in living cells, so the proteins involved are more
likely to retain their functional interacting domains.
Functional cloning
Finally, we consider screening methods that depend
on the full biological activity of the protein. This is
often termed
functional cloning
. In contrast to posi-
tional cloning, described above, functional cloning
is possible in complete ignorance of the whereabouts
of the gene in the genome and requires no prior
knowledge of the nucleotide sequence of the clone or
the amino acid sequence of its product. As long as
the expressed protein is functional and that function
can be exploited to screen an expression library, the
corresponding clone can be identified.
Screening by functional complementation
Functional complementation is the process by which
a particular DNA sequence compensates for a miss-
ing function in a mutant cell, and thus restores the
wild-type phenotype. This can be a very powerful
method of expression cloning, because, if the mutant
cells are non-viable under particular growth condi-
tions, cells carrying the clone of interest can be posi-
tively selected, allowing the corresponding clones to
be isolated.
Ratzkin and Carbon (1977) provide an early
example of how certain eukaryotic genes can be
cloned on the basis of their ability to complement
auxotrophic mutations in
E. coli
. These investigators
inserted fragments of yeast DNA, obtained by mech-
anical shearing, into the plasmid ColEl, using a
homopolymer-tailing procedure. They transformed
E. coli his
B mutants, which are unable to synthesize
histidine, with the recombinant plasmids and plated
the bacteria on minimal medium. In this way, they
selected for complementation of the mutation and
isolated clones carrying an expressed yeast
his
gene.
If the function of the gene is highly conserved, it is
Screening with alternative ligands
As well as DNA and RNA, a whole range of alternat-
ive 'ligands' can be used to identify polypeptides that
specifically bind certain molecules (for example, as
an alternative to south-western screening). Such
techniques are not widely used because they gener-
ally have a low sensitivity and their success depends
